Anti human cd3 ecd

PBMCs were thawed and rested overnight in R10 medium. Cells (5–6 × 10⁶/ml) were stimulated with IE-1 and pp65 peptide pools (2 μg/ml/peptide) in the presence of 1.0 μg/ml anti-CD28/CD49d antibodies for 5 h at 37 °C. Cells were washed with PBS and followed by staining with LIVE/DEAD™ fixable violet stain (Invitrogen). Then the cells were washed and stained with 1/75 diluted anti-human CD3 ECD (Cat: IM2705U, Beckman Coulter). Immediately after staining, single cells gated on live CD3⁺ lymphocytes were sorted on a BD Aria II SORP cytometer. The cells were used for single-cell sequencing within 2 h after sorting.

PBMCs from patients with SLE or RA and HC were collected and analyzed immediately for the molecular phenotypes of lymphocytes. The antibodies used for the surface marker analysis include anti-human CD3-ECD (Beckman Coulter, USA), CD4-Percp-Cy5.5, CD8-PE-Cy7, PD1-FITC, TIGIT-APC, ICOS-PE, TIM3-BV421, CD19-V500, CD3-FITC, CD8-V500, CD25-APC, HLA-DR- PE-Cy7, CD69-V450, and CD127-PE (BD Biosciences, USA). Briefly, 50 μl of cells was incubated with appropriate antibodies on ice in the dark for 30 min, followed by washing in PBS. All the samples were analyzed with a Navios flow cytometer (Beckman Coulter) and Kaluza analysis software (Beckman Coulter).